Cytokines are proteins which are produced during the effector phases of natural and specific immunity and serve to mediate and regulate immune and inflammatory responses. Cytokines, like other polypeptide hormones, initiate their action by binding to specific receptors on the surface of target cells. One of the most well known families of cytokines are the interleukins which mediate natural immunity. For a detailed description of the structure and function of the interleukins, see Abbas et al. Cellular and Molecular Immunology, W. B. Saunders Company, Philadelphia, pp. 225–243, 1991.
IL-3 acts on numerous target cells within the hematopoietic system. A detailed review of the structure and function of this cytokine may be found in The Cytokine Handbook, Third Edition, Thomson, A. Ed., Academic Press, San Diego, Calif., 1998. IL-3 is a glycoprotein having broad structural similarities with other interleukins and hematopoietic growth factors. Murine IL-3 contains 140 amino acids, while human IL-3 contains 133 amino acids. The amino acid sequences of mouse and human IL-3 exhibit only 30% identity, reflecting the lack of cross-species biologic activity (Yang et al., Cell 147:3–10, 1986).
IL-3 has the broadest target specificity of any of the hematopoietic growth factors. The range of target cells includes progenitor cells of every lineage derived from the pluripotential hematopoietic stem cells. Thus, IL-3 can stimulate the generation and differentiation of macrophages, neutrophils, eosinophils, basophils, mast cells, megakaryocytes and erythroid cells. In vitro, hematopoietic stem and progenitor cells rapidly die if cultured in tissue culture medium alone. Like other hematopoietic growth factors, IL-3 prevents death by apoptosis and promotes survival in vitro (Williams et al., Nature 343:76–79, 1990). When deprived of IL-3, IL-3-dependent cells undergo apoptosis (Williams et al., supra.).
The subcutaneous administration of 2000 ED50 units of IL-3 three times a day for three days resulted in an increase in splenic weight and in the number of mast cells and the progenitors of mast cells, neutrophils and macrophages (Schrader et al., Immune Regulation by Characterized Polypeptides, Goldstein, G. et al., eds., Liss, New York, pp. 475–484, 1986). The administration of human IL-3 to primates and humans resulted in similar effects to those seen in mice (Donahue et al., Science 241:1820–1823, 1988; Mayer et al., Blood 74:613–621, 1989). In cynomolgus monkeys, IL-3 induced extramedullary hematopoiesis at sites of subcutaneous injection (Khan et al., Toxicol. Pathol. 24:391–397, 1996). IL-3 may have particular utility in stimulating platelet production (Ganser et al., Blood 76:666–676, 1990). In addition, clinical trials suggest that sequential administration of IL-3 and G-CSF or GM-CSF may provide optimal stimulation of myelopoiesis (Lemoli et al., J. Clin. Oncol. 14:3018–3025, 1996.
Neurotrophins and neurotrophic factors are proteins or peptides capable of affecting the survival, target innervation and/or function of neuronal cell populations (Barde, Neuron, 2:1525–1534, 1989). The efficacy of neurotrophins both in vivo and in vitro has been well documented. For example, ciliary neurotrophic factor (CNTF) promotes survival of chicken embryo ciliary ganglia in vitro and supports survival of cultured sympathetic, sensory and spinal motor neurons (Ip et al., J. Physiol. Paris, 85:123–130, 1991).
A major obstacle to the in vivo therapeutic use of peptides is their susceptibility to proteolytic degradation. The half-life of intravenously injected IL-3 is short, being on the order of only 40 minutes (Crapper et al., Immunology 53:33–42, 1984). Retro-inverso peptides are isomers of linear peptides in which the direction of the sequence is reversed (retro) and the chirality, D or L, of each amino acid is inverted (inverso). There are also partially modified retro-inverso isomers of linear peptides in which only some of the peptide bonds are reversed and the chirality of the amino acid residues in the reversed portion is inverted. The major advantage of such peptides is their enhanced activity in vivo due to improved resistance to proteolytic degradation (For review, see Chorev et al., Trends Biotech., 13:438–445, 1995). Although such retro-inverso analogs exhibit increased metabolic stability, their biological activity is often greatly compromised (Guichard et al., Proc. Natl. Acad. Sci. U.S.A., 91:9765–9769, 1994). For example, Richman et al. (J. Peptide Protein Res., 25:648–662) determined that analogs of linear and cyclic leu-enkephalin modified at the Gly3-Phe4 amide bond had activities ranging from 6%–14% of native leu-enkephalin. Chorev et al., (ibid.) showed that retro-inversion of a peptide which inhibits binding of vitronectin to its receptor resulted in one peptide which was less potent than the parent isomer by a factor of 50,000, and another peptide which was 4,000 fold more potent than the parent cyclic peptide. Guichard et al. (TIBTECH 14, 1996), teach that retro-inverso (all-D-retro) antigenic mimicry may only occur with peptides in random coil, loop or cyclic conformations. In the case of “helical” peptide, adequate functional mimicry would be expected only if the helicity was, in fact, absent under the solvent conditions used for assessing antigenic mimicry.
There is a need for IL-3-derived and neurotrophic peptides exhibiting increased metabolic stability while retaining biological activity. The present invention addresses this need.